CN112708566A - Optimized culture medium and culture method for producing extracellular polymer by aspergillus niger - Google Patents

Optimized culture medium and culture method for producing extracellular polymer by aspergillus niger Download PDF

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CN112708566A
CN112708566A CN202110095946.0A CN202110095946A CN112708566A CN 112708566 A CN112708566 A CN 112708566A CN 202110095946 A CN202110095946 A CN 202110095946A CN 112708566 A CN112708566 A CN 112708566A
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ecp
aspergillus niger
culture
culture medium
fermentation
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刘雪
李林
徐其静
罗增明
赵宇
方迪
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Southwest Forestry University
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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor
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    • C12P1/02Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi

Abstract

The invention belongs to the technical field of culture for producing extracellular polymers by aspergillus niger, and discloses an optimized culture medium and a culture method for producing extracellular polymers (ECP) by aspergillus niger, wherein the optimized culture method for producing the extracellular polymers by the aspergillus niger comprises the following steps: selecting a Aspergillus niger strain with high ECP yield as an experimental material, preparing spore suspension, and carrying out strain fermentation culture; extracting, purifying and analyzing the quantity, chemical components and structures of ECP generated in different growth stages, and exploring the complexation and mechanism of the ECP and heavy metal ions; the culture conditions for producing ECP are optimized by a response surface method. And (3) washing bacterial colonies on the surface of the plate culture medium by using quantitative sterile water on a sterile operating platform, transferring the eluted spores into a sterilized conical flask, adding a small amount of sodium dodecyl sulfate, and shaking uniformly to obtain a spore suspension. The invention provides important reference for further understanding the migration and transformation process of heavy metals in water environment and further disclosing the mechanism of heavy metal adsorption of the biological membrane.

Description

Optimized culture medium and culture method for producing extracellular polymer by aspergillus niger
Technical Field
The invention belongs to the technical field of culture for producing extracellular polymers by aspergillus niger, and particularly relates to an optimized culture medium and a culture method for producing extracellular polymers by aspergillus niger.
Background
At present, Aspergillus niger A80 is a strain of fungal microorganism isolated from heavy metal contaminated soil in certain areas in laboratories. The optimal growth temperature of the strain is 28-30 ℃, extracellular polysaccharide can be secreted in a large amount, and the strain has the advantages of short production period, easily controlled growth conditions and the like. The extracellular polymer of the fungus contains extracellular polysaccharide, protein, fat, oxalic acid, melanin and other substances, and the substances play an important role in the micro-action mechanisms of surface adsorption, complexation, oxalate micro-precipitation, extracellular reduction and the like of heavy metals. The single factor experiment examines the influence of different carbon sources and nitrogen sources on the growth state of the thalli and the yield of the ECP, optimizes the components of an ECP production culture medium, establishes an ECP production model and improves the ECP yield. The microorganism ECP has strong binding capacity to heavy metals, but the interaction mechanism of the ECP and the heavy metals is different due to the difference of components of the ECP of different microorganisms. The research at home and abroad has been mainly focused on the aspect of bacterial ECP, but less relates to the research of fungal ECP. However, studies have shown that fungal cells also exhibit better adsorption capacity for heavy metals, such as rhizogenes, aspergillus fumigatus, and the like. Fungi are easy to culture and can be obtained from many industrial by-products or wastes, and can be used as good biological adsorption materials. A large amount of microbial thalli discharged from industrial fermentation plants and wastewater treatment plants every year are biological adsorbents with potential application values, and the biological adsorbents are used for heavy metal adsorption treatment, so that the treatment problem of heavy metal pollution can be solved while waste is utilized. The biological adsorption technology has attracted general attention in the field of heavy metal polluted wastewater treatment, but the technology is only in an experimental stage at present. At present, domestic researches on biological heavy metal adsorption mainly discuss the influence of various factors on the adsorption process, and the researches on the adsorption mechanism are less. Therefore, aiming at the screened Aspergillus niger fungus, the good complexation of ECP to heavy metal is utilized to treat and repair the heavy metal wastewater pollution, and the micro-molecular action mechanism of the ECP to the heavy metal is discussed by researching the extraction method, yield influence factors, composition and structure of the ECP, so that the method provides technical and theoretical support for further engineering application of the technology and bioremediation of the heavy metal polluted wastewater, obtains the win-win effect of sewage waste resource utilization and environmental ecological remediation, and has important theoretical significance and wide application prospect.
Extracellular polymeric component structure and factors affecting yield
Chemical composition and structure
ECP is a high molecular polymer secreted by microorganisms, and has a complex composition, and its composition and structure exhibit different characteristics depending on the kind of microorganisms. ECPs can be broadly classified into the following groups according to their chemical composition: polysaccharides, such as chitosan and cellulose heteropolysaccharides; polyamides, such as proteins and polyamino acids; nucleic acids such as deoxyribonucleic acid and ribonucleic acid; a polythioester; polyphenols, such as humic acid and lignin; polyisoprene, such as natural rubber or gutta percha. Of these, polysaccharides are most common, followed by proteins.
Environmental influence factor
Environmental factors that affect microbial growth and ECP production include nutrient conditions of the culture medium and physical environmental conditions of the culture. The nutrient conditions of the culture medium mainly comprise: carbon sources, nitrogen sources and other nutrients. Culturing physical environmental conditions comprises: system pH value, temperature, culture time, shaking and stirring speed and the like.
Nutrient conditions
Carbon sources have a significant effect on the distribution of microbial ECP and its yield. The secreted ECP is mainly distributed on the cell surface when the strain R.erythropolisn takes n-pentadecane as a carbon source; whereas in the case of glucose as carbon source, most of the ECP produced is present in the fermentation broth. In addition, when ECP is produced using the strain r.erythropolisn, the yield of ECP is higher with n-pentadecane as a carbon source than with glucose as a carbon source. Researches find that for the strain C.glutamicum CCTCCM 201005, in the growth process of the bacteria, glucose, sucrose and fructose can be better utilized, which is beneficial to the synthesis of ECP; while the use of soluble starch, galactose, ethanol, sodium acetate, etc. as single carbon sources is not favorable for the synthesis and secretion of ECP. The nitrogen source also significantly affects the growth, reproduction and metabolism of the microorganism. It has been reported that Aspergillus niger grows well in yeast extract, ammonium chloride, ammonium sulfate, sodium nitrate, beef extract, fish meal, soybean meal, peptone and urea, but only in media with sodium nitrate and urea as nitrogen sources, ECP is synthesized well. In the process of producing ECP using strain r.erythropolis, it was found that when ammonium sulfate and urea were selected as inorganic nitrogen sources and casein hydrolysate and yeast extract were selected as organic nitrogen sources, the strain grew well and the ECP synthesis was promoted, but when some inorganic nitrogen compounds (e.g., ammonium sulfate, ammonium chloride, ammonium nitrate) were used as nitrogen sources, the strain mycelia hardly grew and ECP synthesis was not efficient. Bacillus licheniformis CCRC12826 showed an ECP yield of up to 13.9g/L in a medium with ammonium chloride as a nitrogen source, but the strain was not able to efficiently synthesize ECP in a medium with ammonium nitrate, peptone and ammonium sulfate as nitrogen sources.
The environmental conditions, the pH of the liquid fermentation system, are the main factors for efficient microbial growth and ECP synthesis. The increase or decrease of the pH value of the culture medium can influence the transfer of electrons in the metabolic process of the microorganism, thereby influencing the synthesis and secretion of the bacterial ECP. Different ECP producing bacteria have different preferred pH ranges. Researches show that in the pH range of 5-7, Aspergillus niger bacteria can well grow, fermentation liquor is viscous, and the ECP yield is high; the highest yield of ECP was achieved at an initial pH of 6 in the fermentation medium. Whereas strain c. xerosis is suitable for the synthesis of ECP under lower pH environmental conditions. The optimal H value range of ECP produced by Pseudomonas sp.GX4-1 is pH 7-9, the initial pH of fermentation is too high or too low, which is not beneficial to the growth of thalli and the synthesis of ECP, and the bacteria can hardly synthesize ECP particularly under the acidic condition. Furthermore, the optimal pH range for the synthesis of ECP is not completely consistent with the optimal pH range for the growth of the strain. The optimum pH value for the growth of the Paenibacillus polymyxa GA1 thallus is 7.0, the optimum initial pH value for synthesizing ECP is 6.5, and the thallus growth is not favored by the acid or alkali environment.
Temperature is another important factor that affects ECP yield. The ECP producing bacteria realize the mass synthesis of ECP under the respective optimal temperature conditions. It was found that various bacteria (Pseudomonas sp. GX4-1, Bacillus sp.DP-152, Alc. latus and Citrobacter sp.TKF04) can efficiently synthesize ECP at 30 ℃. The optimal synthetic ECP temperature range for Aspergillus niger production is 30-34 ℃; the temperature for the best ECP production of the strain Zoogloea MP6 is 20 ℃; BY-29, this temperature was 37 ℃ for the strain Enterobacter sp.
Aeration is also one of the factors that affect ECP production. The ventilation is realized by controlling the liquid loading amount or the rotating speed of the shaking table. The liquid loading amount is large, the mass transfer rate is slow, and the concentration of dissolved oxygen is low; the shaking table has high oscillation speed, the dissolved oxygen is supplemented in time, and the concentration of the dissolved oxygen in the fermentation liquor is high. When Bacillus megarium A25 was cultured in a 250mL Erlenmeyer flask, it was found that ECP synthesis was not detected in the fermentation broth when the liquid content exceeded 150 mL. In the process of culturing the strain Pseudomonas putida B6, when the shaking speed of a shaking table is 80rpm, thalli in fermentation liquor are aggregated to form clusters, so that the full contact of the thalli and the culture solution is influenced, the utilization of nutrient substances is not facilitated, and the growth of the thalli and the synthesis of ECP are influenced; however, too large ventilation is not good for Pseudomonas putida B6 to secrete ECP, and the optimum rotating speed of the shaking table is 120-160 rpm. The amount of inoculum also affects the synthesis of ECP. The inoculation amount is too large, the initial concentration of thalli in the fermentation liquor is too high, and the growth and the propagation of the thalli cause the excessive consumption of nutrient substances of the fermentation liquor, so that the ECP is not beneficial to synthesis; the inoculation amount is too small, the thallus concentration of the fermentation liquor is too low, the culture period of the strain is prolonged, and the yield of ECP is reduced.
The traditional optimization method is a single-factor experiment method, namely, the optimization method only changes one value range of the influence factor in each experiment. The method is simple to operate, does not need a theoretical basis in mathematical statistics, and is one of common optimization methods, wherein results are visually presented by a chart. However, when the influence of more factors is examined, a large number of experiments and a long experiment period are needed, and in the experiment process, errors of conditions and operation are inevitably generated, and mutual influence among the factors is not considered, so that the result is unreliable or an error conclusion is drawn. It is not suitable to evaluate the influence of multiple factors.
The orthogonal experimental design method is widely applied to condition optimization. The same-factor and same-level experiment shows that the experiment frequency of orthogonal experiment design is obviously less than that of a single-factor experiment, the experiment shows obvious superiority, the influence significance of each factor on the experiment result can be obtained through variance analysis, and meanwhile the interaction among the influencing factors is considered. Orthogonal test design also has the defect that when the interaction among factors is taken as a factor for investigation, the total test times can be greatly increased, so that the experimental workload is obviously increased. Under the condition of long single experiment period, a large amount of manpower, material resources and time are needed.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) when the influence of more factors is inspected, a large number of experiments and a long experiment period are needed, in the experiment process, the single-factor experiment method inevitably generates errors of conditions and operation, and mutual influence among the factors is not considered, so that the result is unreliable or an error conclusion is obtained. It is not suitable to evaluate the influence of multiple factors.
(2) In the orthogonal test design method, when the interaction among the factors is taken as a factor for investigation, the total test times can be greatly increased, so that the test workload is obviously increased. Under the condition of long single experiment period, a large amount of manpower, material resources and time are needed.
The difficulty in solving the above problems and defects is: the biological adsorption technology has attracted general attention in the field of heavy metal polluted wastewater treatment, but the technology is only in an experimental stage at present. At present, domestic researches on biological heavy metal adsorption mainly discuss the influence of various factors on the adsorption process, and the researches on the adsorption mechanism are less.
The significance of solving the problems and the defects is as follows: the microorganism ECP has strong binding capacity to heavy metals, but the interaction mechanism of the ECP and the heavy metals is different due to the difference of components of the ECP of different microorganisms. The research at home and abroad has been mainly focused on the aspect of bacterial ECP, but less relates to the research of fungal ECP. However, studies have shown that fungal cells also exhibit better adsorption capacity for heavy metals, such as Rhizopus arrhizus, Aspergillus fumigatus. Fungi are easy to culture and can be obtained from many industrial by-products or wastes, and can be used as good biological adsorption materials. A large amount of microbial thalli discharged from industrial fermentation plants and wastewater treatment plants every year are biological adsorbents with potential application values, and the biological adsorbents are used for heavy metal adsorption treatment, so that the treatment problem of heavy metal pollution can be solved while waste is utilized.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an optimized culture medium and a culture method for producing extracellular polymeric substances by Aspergillus niger.
The invention is realized in such a way that an aspergillus niger optimized culture medium for producing extracellular polymers comprises: plate culture medium-PDA culture medium, fermentation culture medium;
plate medium-PDA medium: 200g/L of potato, 15-20 g/L of agar, 20g/L of glucose, natural pH and autoclaving at 121 ℃ for 20 min;
fermentation medium: NaNO3 1.5g/L,KH2PO4 0.5g/L,KCl 0.025g/L,MgSO4·7H20.025g/L of O, 100g/L of sucrose, 1.6 of yeast extract, natural pH and autoclaving at 121 ℃ for 20 min.
Further, peeling and cleaning potato in the plate culture medium-PDA culture medium, weighing 200g, cutting into small blocks, adding water, boiling until the potato is soft, filtering with eight layers of gauze, adding agar and glucose in calculated amount into the filtrate, uniformly mixing, supplementing to 1000mL with distilled water, sterilizing at 121 ℃ for 20min, and making into slant or plate for strain preservation.
Another objective of the present invention is to provide an optimized culture method for producing extracellular polymeric substance by aspergillus niger, which comprises:
step one, selecting a strain of Aspergillus niger A80 with high ECP yield as an experimental material, preparing spore suspension, and carrying out strain fermentation culture;
extracting, purifying and analyzing the quantity, chemical components and structures of ECP generated in different growth stages, and exploring the complexation and mechanism of ECP and heavy metal ions;
and step three, optimizing the culture conditions for producing ECP by A.niger A80 by a response surface method.
Further, in the first step, the preparation process of the spore suspension comprises the following steps:
and (3) washing the bacterial colony on the surface of the plate culture medium with quantitative sterile water on a sterile operating platform, transferring the eluted spores into a sterilized conical flask, repeating the steps for several times, adding a small amount of 0.02% (w/v) sodium dodecyl sulfate, and shaking uniformly to obtain a high-concentration spore suspension, wherein the concentration of the spores is about 107/mL.
Further, in the first step, the strain fermentation culture comprises the following steps:
300mL of fermentation medium was placed in a 500mL conical flask, 3mL of spore suspension was inoculated after sterilization, and the mixture was placed at 25 ℃ in a shaker at 150rpm for fermentation for a predetermined period of time.
Further, in the second step, the method for extracting the coarse ECP comprises:
the pH value of the system is increased by adding strong alkali sodium hydroxide into the thallus fermentation liquor, so that on one hand, the dissociation of acid groups in ECP and the mutual repulsion between ECP with negative electricity per se are promoted; on the other hand, the solubility of ECP in water is improved, thereby promoting the entry of ECP into the water phase.
Further, in the second step, the ECP purification method includes:
concentrating the supernatant, removing protein by Seveag method, sequentially adding chloroform and n-butanol into the supernatant, shaking the mixed solution sufficiently, centrifuging at 8000rpm for 10min at 4 deg.C, removing middle denatured protein and lower layer chloroform, pouring out the supernatant, and repeating the above steps until no obvious denatured protein appears in the middle;
the ECP solution after deproteinization treatment is subjected to fractional precipitation, freezing and centrifugation by adopting ethanol to remove nucleic acid, firstly, 25% of glacial ethanol is used for precipitation to remove nucleic acid, supernatant fluid is collected by centrifugation, 75% of glacial ethanol is reprecipitated, precipitate is sequentially washed by absolute ethyl alcohol and acetone and then dissolved in distilled water again, the precipitate is placed in a dialysis bag of 8000-14000 Da at the temperature of 4 ℃ for dialysis for 48h, and the dialysis solution in dialysis is collected and freeze-dried to obtain the pure ECP.
Further, among the chloroform and n-butanol, chloroform/n-butanol is 5:1, V/V.
Further, the conditions for removing nucleic acid by 25% glacial ethanol precipitation are as follows: 24 hours at 4 ℃;
the conditions for 75% glacial ethanol reprecipitation were: 4 ℃ and 24 h.
Further, in the third step, the specific process is as follows:
fitting the experimental data by using a response surface model to obtain each parameter in the model;
performing simple character analysis on the multivariate function to determine an extreme point and a value of an independent variable corresponding to the extreme point;
and carrying out experiments according to the optimal conditions, verifying the reliability of the model, and determining the final optimization result.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention aims to provide important reference for further understanding the migration and transformation process of heavy metals in water environment and further disclosing the mechanism of heavy metal adsorption of a biological membrane. 4 influencing factors of the RSM method based on central combinatorial design on the growth of a. niger a 80: the optimization research of lactose adding amount, yeast extract adding amount, culture medium pH and culture period shows that: when the lactose adding amount, the yeast extract adding amount, the culture medium pH and the culture period are respectively 120g/L, 2.0g/L, 6.5 and 116h, the yield of the ECP is the maximum and reaches 339TOC mg/L. The yield of the optimized ECP is 323TOC mg/L, the error of the model fitting result is within 10 percent, and the obtained model is reliable.
Meanwhile, single factor experiments of an optimal carbon source show that Aspergillus niger A80 can well grow in a culture medium with glucose, sucrose and lactose as single carbon sources, and soluble starch is not easy to utilize by the strain. When sucrose is used as a single carbon source, the growth amount of the mycelium pellets is the highest; when lactose is used as a carbon source, the growth speed of the mycelium pellets is the fastest. From the comprehensive consideration of the growing speed of mycelium pellets and the yield of ECP, lactose can be selected as a long-acting carbon source in the fermentation liquid process of Aspergillus niger A80, and sucrose can be selected as a short-acting carbon source from the consideration of raw material sources and industrial cost. Through single factor experiments of an optimal nitrogen source, the aspergillus niger A80 can grow in a culture medium with ammonium sulfate, ammonium chloride, beef extract, peptone and yeast extract as single nitrogen sources. When the yeast extract is used as a single nitrogen source, the growth amount of the mycelium pellets is the highest, the growth speed of the mycelium pellets is the fastest, and the yeast extract is selected as the nitrogen source of the culture medium in the process of the Aspergillus niger A80 fermentation liquid. Four influencing factors are optimized by 30 experiments by applying central combination design. The optimization result shows that when the lactose adding amount, the yeast extract adding amount, the culture medium pH and the culture period are respectively 120g/L, 2.0g/L, 6.5 h and 116h, the model predicts that the ECP yield is the highest and reaches 339TOC mg/L. The yield of the optimized ECP is 323mg/L, and the error of the model fitting result is within 10 percent, which indicates that the obtained model is reliable.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of the optimized culture method for producing extracellular polymeric substances by Aspergillus niger according to the embodiment of the present invention.
FIG. 2 is a schematic diagram of the colony morphology of Aspergillus niger A80 provided in the examples of the present invention.
In FIG. (A), PDA medium; in FIG. B, the microscope (10X 10).
Fig. 3 is a flowchart of a coarse ECP extraction method according to an embodiment of the present invention.
FIG. 4 is a schematic diagram of A80 mycelial pellets in a liquid medium provided by an embodiment of the invention.
FIG. 6h, FIG. 12h, FIG. 24h, FIG. 36h, FIG. 48h and FIG. 60F.
FIG. 5 is a schematic diagram of A80 cells in the solid medium according to the present invention.
FIG. 6h, FIG. 12h, FIG. 24h, FIG. 36h, FIG. 48h and FIG. 60F.
FIG. 6 is a schematic illustration of ECP precipitated by glacial ethanol provided by an embodiment of the invention.
FIG. 3 (A), FIG. 6 (B), and FIG. 10 (C).
Figure 7 is a schematic representation of the ECP after freeze-drying as provided by an embodiment of the invention.
FIG. 3 (A), FIG. 6 (B), and FIG. 10 (C).
FIG. 8 is a SEM image of 3d ECP after freeze-drying as provided in the examples herein.
FIG. 9 is a SEM representation of 6d ECP after freeze-drying as provided in the examples herein.
FIG. 10 is a SEM representation of a freeze-dried 10d ECP provided by an embodiment of the present invention.
FIG. 11 is a schematic diagram of the adsorption isotherms of ECP on methylene blue at different times according to an embodiment of the present invention.
FIG. 12 is a Languuir adsorption isotherm of ECP on methylene blue at various times as provided in an example of the present invention.
FIG. 13 is a graphical representation of the cross-response between lactose dosage and yeast cream dosage provided by an embodiment of the present invention.
FIG. 14 is a graph of the interaction response of lactose dosage with pH of the medium provided by the example of the present invention.
FIG. 15 is a graphical representation of the interaction of lactose dosage with incubation period provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides an optimized culture medium and a culture method for producing extracellular polymeric substances by Aspergillus niger, and the invention is described in detail below with reference to the attached drawings.
The embodiment of the invention provides an optimized culture medium for producing extracellular polymeric substances by Aspergillus niger, which comprises:
(1) plate medium-PDA medium (g/L): 200 parts of potato, 15-20 parts of agar and 20 parts of glucose, wherein the pH is natural, and the potato is sterilized at 121 ℃ for 20 min. Peeling and cleaning potato, weighing 200g, cutting into small pieces, adding water, boiling until the potato is soft, filtering with eight layers of gauze, adding agar and glucose in calculated amount into filtrate, mixing uniformly, supplementing to 1000mL with distilled water, sterilizing at 121 deg.C for 20min, and making into slant or plate, wherein the culture medium is used for strain preservation.
(2) Fermentation medium (g/L): NaNO3 1.5,KH2PO4 0.5,KCl 0.025,MgSO4·7H2O0.025, sucrose 100, yeast extract 1.6, natural pH, and autoclaving at 121 deg.C for 20 min.
As shown in fig. 1, the optimized culture method for producing extracellular polymeric substance by aspergillus niger provided in the embodiment of the present invention includes:
s101: a strain of Aspergillus niger A80 with high ECP yield is selected as an experimental material, spore suspension is prepared, and strain fermentation culture is carried out.
S102: extracting, purifying and analyzing the quantity, chemical components and structures of ECP generated in different growth stages, and exploring the complexation and mechanism of ECP and heavy metal ions.
S103: culture conditions for a. niger a80 production of ECP were optimized by the response surface method.
In S101 provided by the embodiment of the present invention, the preparation process of the spore suspension is:
and (3) washing the bacterial colony on the surface of the plate culture medium with quantitative sterile water on a sterile operating platform, transferring the eluted spores into a sterilized conical flask, repeating the steps for several times, adding a small amount of 0.02% (w/v) sodium dodecyl sulfate, and shaking uniformly to obtain a high-concentration spore suspension, wherein the concentration of the spores is about 107/mL.
In S101 provided by the embodiments of the present invention, the strain fermentation culture is:
300mL of fermentation medium was placed in a 500mL conical flask, 3mL of spore suspension was inoculated after sterilization, and the mixture was placed at 25 ℃ in a shaker at 150rpm for fermentation for a predetermined period of time.
In S102 provided in the embodiment of the present invention, the method for extracting coarse ECP includes:
the pH value of the system is increased by adding strong alkali sodium hydroxide into the thallus fermentation liquor, so that on one hand, the dissociation of acid groups in ECP and the mutual repulsion between ECP with negative electricity per se are promoted; on the other hand, the solubility of ECP in water is improved, thereby promoting the entry of ECP into the water phase.
In S102 provided in an embodiment of the present invention, the ECP purification method includes:
concentrating the supernatant, removing protein by Seveag method, sequentially adding chloroform and n-butanol (chloroform: n-butanol is 5:1, V/V) into the supernatant, shaking the mixed solution sufficiently, centrifuging at 8000rpm for 10min at 4 deg.C, removing middle denatured protein and lower layer chloroform, pouring out the supernatant, repeating the above process for several times until no obvious denatured protein appears in the middle;
the ECP solution after deproteinization is subjected to fractional precipitation, freezing and centrifugation by adopting ethanol to remove nucleic acid, firstly, 25% of glacial ethanol is used for precipitation to remove the nucleic acid (4 ℃ for 24 hours), supernatant is collected by centrifugation, 75% of glacial ethanol is used for reprecipitation (4 ℃ for 24 hours), precipitates are sequentially washed by absolute ethyl alcohol and acetone and then are dissolved in distilled water again, the precipitates are placed in a dialysis bag of 8000-14000 Da for dialysis at 4 ℃ for 48 hours, and the dialysis solution is collected, cooled and dried to obtain the ECP pure product.
In S103 provided by the embodiment of the present invention, the specific process is as follows:
fitting the experimental data by using a response surface model to obtain each parameter in the model; performing simple character analysis on the multivariate function to determine an extreme point and a value of an independent variable corresponding to the extreme point; and carrying out experiments according to the optimal conditions, verifying the reliability of the model, and determining the final optimization result.
The technical solution of the present invention will be further described with reference to specific experiments.
1.1 extracellular Polymer component Structure and factors affecting yield
1.1.1 chemical composition and Structure
ECP is a high molecular polymer secreted by microorganisms, and has a complex composition, and its composition and structure exhibit different characteristics depending on the kind of microorganisms. ECPs can be broadly classified into the following groups according to their chemical composition: polysaccharides, such as chitosan and cellulose heteropolysaccharides; polyamides, such as proteins and polyamino acids; nucleic acids such as deoxyribonucleic acid and ribonucleic acid; a polythioester; polyphenols, such as humic acid and lignin; polyisoprene, such as natural rubber or gutta percha. Of these, polysaccharides are most common, followed by proteins.
1.1.2 environmental influences
Environmental factors that affect microbial growth and ECP production include nutrient conditions of the culture medium and physical environmental conditions of the culture. The nutrient conditions of the culture medium mainly comprise: carbon sources, nitrogen sources and other nutrients. Culturing physical environmental conditions comprises: system pH value, temperature, culture time, shaking and stirring speed and the like.
The nutrient conditions, carbon sources, have a significant effect on the distribution of microbial ECP and its yield. The secreted ECP is mainly distributed on the cell surface when the strain R.erythropolisn takes n-pentadecane as a carbon source; whereas in the case of glucose as carbon source, most of the ECP produced is present in the fermentation broth. In addition, when ECP is produced using the strain r.erythropolisn, the yield of ECP is higher with n-pentadecane as a carbon source than with glucose as a carbon source. Researches find that for the strain C.glutamicum CCTCCM 201005, in the growth process of the bacteria, glucose, sucrose and fructose can be better utilized, which is beneficial to the synthesis of ECP; while the use of soluble starch, galactose, ethanol, sodium acetate, etc. as single carbon sources is not favorable for the synthesis and secretion of ECP.
The nitrogen source also significantly affects the growth, reproduction and metabolism of the microorganism. It has been reported that Aspergillus niger grows well in yeast extract, ammonium chloride, ammonium sulfate, sodium nitrate, beef extract, fish meal, soybean meal, peptone and urea, but only in media with sodium nitrate and urea as nitrogen sources, ECP is synthesized well. In the process of producing ECP using strain r.erythropolis, it was found that when ammonium sulfate and urea were selected as inorganic nitrogen sources and casein hydrolysate and yeast extract were selected as organic nitrogen sources, the strain grew well and the ECP synthesis was promoted, but when some inorganic nitrogen compounds (e.g., ammonium sulfate, ammonium chloride, ammonium nitrate) were used as nitrogen sources, the strain mycelia hardly grew and ECP synthesis was not efficient. Bacillus licheniformis CCRC12826 showed an ECP yield of up to 13.9g/L in a medium with ammonium chloride as a nitrogen source, but the strain was not able to efficiently synthesize ECP in a medium with ammonium nitrate, peptone and ammonium sulfate as nitrogen sources.
The environmental conditions, the pH of the liquid fermentation system, are the main factors for efficient microbial growth and ECP synthesis. The increase or decrease of the pH value of the culture medium can influence the transfer of electrons in the metabolic process of the microorganism, thereby influencing the synthesis and secretion of the bacterial ECP. Different ECP producing bacteria have different preferred pH ranges. Researches show that in the pH range of 5-7, Aspergillus niger bacteria can well grow, fermentation liquor is viscous, and the ECP yield is high; the highest yield of ECP was achieved at an initial pH of 6 in the fermentation medium. While strain c. xerosis is suitable for ECP synthesis in very low pH environments. The optimal pH value range of ECP produced by Pseudomonas sp.GX4-1 is pH 7-9, the initial pH value of fermentation is too high or too low, which is not beneficial to the growth of thalli and the synthesis of ECP, and the bacteria can hardly synthesize ECP particularly under the acidic condition. Furthermore, the optimal pH range for the synthesis of ECP is not completely consistent with the optimal pH range for the growth of the strain. The optimum pH value for the growth of the Paenibacillus polymyxa GA1 thallus is 7.0, the optimum initial pH value for synthesizing ECP is 6.5, and the thallus growth is not favored by the acid or alkali environment.
Temperature is another important factor that affects ECP yield. The ECP producing bacteria realize the mass synthesis of ECP under the respective optimal temperature conditions. It was found that various bacteria (Pseudomonas sp. GX4-1, Bacillus sp.DP-152, Alc. latus and Citrobacter sp.TKF04) can efficiently synthesize ECP at 30 ℃. The optimal synthetic ECP temperature range for Aspergillus niger production is 30-34 ℃; the temperature for the best ECP production of the strain Zoogloea MP6 is 20 ℃; BY-29, this temperature was 37 ℃ for the strain Enterobacter sp.
Aeration is also one of the factors that affect ECP production. The ventilation is realized by controlling the liquid loading amount or the rotating speed of the shaking table. The liquid loading amount is large, the mass transfer rate is slow, and the concentration of dissolved oxygen is low; the shaking table has high oscillation speed, the dissolved oxygen is supplemented in time, and the concentration of the dissolved oxygen in the fermentation liquor is high. When Bacillus megarium A25 was cultured in a 250mL Erlenmeyer flask, it was found that ECP synthesis was not detected in the fermentation broth when the liquid content exceeded 150 mL. In the process of culturing the strain Pseudomonas putida B6, when the shaking speed of a shaking table is 80rpm, thalli in fermentation liquor are aggregated to form clusters, so that the full contact of the thalli and the culture solution is influenced, the utilization of nutrient substances is not facilitated, and the growth of the thalli and the synthesis of ECP are influenced; however, too large ventilation is not good for Pseudomonas putida B6 to secrete ECP, and the optimum rotating speed of the shaking table is 120-160 rpm.
The amount of inoculum also affects the synthesis of ECP. The inoculation amount is too large, the initial concentration of thalli in the fermentation liquor is too high, and the growth and the propagation of the thalli cause the excessive consumption of nutrient substances of the fermentation liquor, so that the ECP is not beneficial to synthesis; the inoculation amount is too small, the thallus concentration of the fermentation liquor is too low, the culture period of the strain is prolonged, and the yield of ECP is reduced.
1.2 Medium composition optimization
Factors influencing the growth state of thalli and the yield of products in the fermentation process are very complex, and comprise a series of factors such as a culture medium carbon source, a nitrogen source, a pH value, a culture temperature, a shaking table rotating speed, a liquid loading amount, an inoculation amount, a seed age and the like, the factors are not always independently acted on the fermentation process, and various factors usually have interaction. The optimization of the components of the microbial fermentation medium mainly comprises the following steps: 1) confirmation of all influence factors; 2) screening the significant influence factors; 3) selecting and designing an optimization strategy; 4) carrying out mathematical or statistical analysis on the experimental result to determine the optimal condition; 5) experimental verification of optimal conditions.
The traditional optimization method is a single-factor experiment method, namely, the optimization method only changes one value range of the influence factor in each experiment. The method is simple to operate, does not need a theoretical basis in mathematical statistics, and is one of common optimization methods, wherein results are visually presented by a chart. However, when the influence of more factors is examined, a large number of experiments and a long experiment period are needed, and in the experiment process, errors of conditions and operation are inevitably generated, and mutual influence among the factors is not considered, so that the result is unreliable or an error conclusion is drawn. It is not suitable to evaluate the influence of multiple factors.
The orthogonal experimental design method is widely applied to condition optimization. The same-factor and same-level experiment shows that the experiment frequency of orthogonal experiment design is obviously less than that of a single-factor experiment, the experiment shows obvious superiority, the influence significance of each factor on the experiment result can be obtained through variance analysis, and meanwhile the interaction among the influencing factors is considered. Orthogonal test design also has the defect that when the interaction among factors is taken as a factor for investigation, the total test times can be greatly increased, so that the experimental workload is obviously increased. Under the condition of long single experiment period, a large amount of manpower, material resources and time are needed.
In order to make up for the defects of the traditional single-factor experimental method and the orthogonal experimental design method, a statistical optimization method is adopted for optimizing a large number of biological processes. Among them, the Response Surface Method (RSM) is a comprehensive method for optimization using a large number of biological process factors. RSM is a product of combining a mathematical method and a statistical method, a mathematical model is used for describing the relation between an influence factor of an experiment and a target response value, and the optimal condition can be determined through numerical analysis. The purpose is to optimize the response value.
The Central Composite Design (CCD) is a response surface method which is commonly used internationally. By adopting the method, influence factors influencing the biological process and interaction thereof can be evaluated under limited experiment times, and each factor is optimized to obtain the optimal condition for realizing the maximum response value.
1.3 brief introduction to Aspergillus niger
Aspergillus niger A80 is a fungal microorganism isolated from heavy metal contaminated soil in certain areas in laboratories. The optimal growth temperature of the strain is 28-30 ℃, extracellular polysaccharide can be secreted in a large amount, and the strain has the advantages of short production period, easily controlled growth conditions and the like. The extracellular polymer of the fungus contains extracellular polysaccharide, protein, fat, oxalic acid, melanin and other substances, and the substances play an important role in micro-action mechanisms such as surface adsorption, complexation, oxalate micro-precipitation, extracellular reduction and the like without heavy metal. The Aspergillus niger A80 provided by the embodiment of the invention is Aspergillus niger A80, and the preservation number of the strain is CGMCC 4533. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of West Lu No. 1 of the North Cheng of the Korean-Yang district, Beijing, and the institute of microorganisms of the Chinese academy of sciences, wherein the strain is preserved in 10 days 1 month 2011, and the postal number is 100101; the reference biomaterial is a 80; suggested classification nomenclature: aspergillus niger; the biological material has been received by the depository at 10/1/2011 and registered in a book; according to requirements, the biological material sample is stored for thirty years from 1 month and 10 days 2011, and is stored for five years after receiving a request for providing the biological material sample before the expiration; the viability of the biological material was tested by the depository at 2011, 1/10 days, and the results were survival.
1.4 content and significance of the study
1.4.1 study content; the single factor experiment examines the influence of different carbon sources and nitrogen sources on the growth state of the thalli and the yield of the ECP, optimizes the components of an ECP production culture medium, establishes an ECP production model and improves the ECP yield.
1.4.2 research purposes, theoretical significance and practical application value; the microorganism ECP has strong binding capacity to heavy metals, but the interaction mechanism of the ECP and the heavy metals is different due to the difference of components of the ECP of different microorganisms. The research at home and abroad has been mainly focused on the aspect of bacterial ECP, but less relates to the research of fungal ECP. However, studies have shown that fungal cells also exhibit better adsorption capacity for heavy metals, such as rhizogenes, aspergillus fumigatus, and the like. Fungi are easy to culture and can be obtained from many industrial by-products or wastes, and can be used as good biological adsorption materials. A large amount of microbial thalli discharged from industrial fermentation plants and wastewater treatment plants every year are biological adsorbents with potential application values, and the biological adsorbents are used for heavy metal adsorption treatment, so that the treatment problem of heavy metal pollution can be solved while waste is utilized.
The biological adsorption technology has attracted general attention in the field of heavy metal polluted wastewater treatment, but the technology is only in an experimental stage at present. At present, domestic researches on biological heavy metal adsorption mainly discuss the influence of various factors on the adsorption process, and the researches on the adsorption mechanism are less.
Therefore, aiming at the screened Aspergillus niger fungus, the good complexation of ECP to heavy metal is utilized to treat and repair the heavy metal wastewater pollution, and the micro-molecular action mechanism of the ECP to the heavy metal is discussed by researching the extraction method, yield influence factors, composition and structure of the ECP, so that the method provides technical and theoretical support for further engineering application of the technology and bioremediation of the heavy metal polluted wastewater, obtains the win-win effect of sewage waste resource utilization and environmental ecological remediation, and has important theoretical significance and wide application prospect.
2.1 Experimental reagents
CaCl2(AR),MgCl2(AR),HCI(AR),NaOH(AR),NaNO3(AR),MgSO4(AR),KCl(AR),KH2PO4(AR),NH4Cl(AR),(NH4)2SO4(AR),CdCl2(AR),Pb(NO3)2(AR),ZnSO4(AR),H2SO4(AR), hydrogen peroxide, sucrose, glucose, lactose, soluble starch, yeast extract, methylene blue, ammonium molybdate, ascorbic acid, bovine serum albumin, 1-phenyl-3-methyl-5-pyrazolone (PMP), zinc standard solution, cadmium standard solution, lead standard solution.
2.2 Experimental instruments
The instruments and equipment used in the experiment are shown in Table 1.
TABLE 1 instruments and apparatus
Figure BDA0002914047480000101
2.3 sources of the Strain
The ECP-producing strain used in the experiment is obtained by separating and screening from soil in a flat plate dilution scribing method in a laboratory, the strain is black in a spore region on a PDA culture medium, hypha is white, the colony form is circular villous, the edge is neat, the surface is slightly convex, the hypha has branches and a diaphragm, as shown in figure 2, the colony form of Aspergillus niger A80 is identified as Aspergillus niger named Aspergillus niger A80, the strain preservation number is CGMCC4533, and the strain is hereinafter referred to as Aspergillus niger A80.
2.4 Medium
(1) Plate medium-PDA medium (g/L): 200 parts of potato, 15-20 parts of agar and 20 parts of glucose, wherein the pH is natural, and the potato is sterilized at 121 ℃ for 20 min. Peeling and cleaning potato, weighing 200g, cutting into small pieces, adding water, boiling until the potato is soft, filtering with eight layers of gauze, adding agar and glucose in calculated amount into filtrate, mixing uniformly, supplementing to 1000mL with distilled water, sterilizing at 121 deg.C for 20min, and making into slant or plate, wherein the culture medium is used for strain preservation.
(2) Fermentation medium (g/L): NaNO3 1.5,KH2PO4 0.5,KCl 0.025,MgSO4·7H2O0.025, sucrose 100, yeast extract 1.6, natural pH, and autoclaving at 121 deg.C for 20 min.
2.5 Strain culture and preparation of ECP
2.5.1 preparation of spore suspension
Washing bacterial colony on the surface of the plate culture medium with quantitative sterile water on a sterile operating platform, transferring the eluted spores to a sterilized conical flask, repeating the steps for several times, adding a small amount of 0.02% (w/v) sodium dodecyl sulfate, and shaking uniformly to obtain a high-concentration spore suspension liquid, wherein the concentration of the spores is about 107one/mL.
2.5.2 fermentation culture of the Strain
300mL of fermentation medium was placed in a 500mL conical flask, 3mL of spore suspension was inoculated after sterilization, and the mixture was placed at 25 ℃ in a shaker at 150rpm for fermentation for a predetermined period of time.
2.5.3 extraction of crude ECP
See fig. 3. The pH value of the system is increased by adding strong alkali sodium hydroxide into the thallus fermentation liquor, so that on one hand, the dissociation of acid groups in ECP and the mutual repulsion between ECP with negative electricity per se can be promoted; on the other hand, the solubility of ECP in water can be improved, thereby promoting the entry of ECP into the water phase.
2.5.4 purification of ECP
The concentrated supernatant was obtained as 2.5.3. Concentrating the supernatant, removing protein by Seveag method, sequentially adding chloroform and n-butanol (chloroform: n-butanol is 5:1, V/V) into the supernatant, shaking the mixture sufficiently, centrifuging at 8000rpm for 10min at 4 deg.C, removing middle denatured protein and lower layer chloroform, decanting the supernatant, and repeating the above steps until no middle denatured protein appears. The ECP solution after deproteinization is subjected to fractional precipitation, freezing and centrifugation by adopting ethanol to remove nucleic acid, firstly, 25% of glacial ethanol is used for precipitation to remove the nucleic acid (4 ℃ for 24 hours), supernatant is collected by centrifugation, 75% of glacial ethanol is used for reprecipitation (4 ℃ for 24 hours), precipitates are sequentially washed by absolute ethyl alcohol and acetone and then are dissolved in distilled water again, the precipitates are placed in a dialysis bag of 8000-14000 Da for dialysis at 4 ℃ for 48 hours, and the dialysis solution is collected, cooled and dried to obtain the ECP pure product.
3.1 analysis of physical Properties
3.1.1 mycelial sphere morphological characteristics
The adsorption performance of the thallus suspension of the aspergillus niger A80 in the liquid culture medium on the heavy metal mainly comes from three aspects of effects: 1) in the culture process of the aspergillus niger A80, spores and thalli form numerous small mycelium pellets (figure 4), and the mycelium pellets have certain specific surface area and can adsorb heavy metal ions in liquid; 2) the cell product secreted by the aspergillus niger A80 thallus in the growth process has good adsorption effect on heavy metal ions; 3) the cell wall of the aspergillus niger A80 contains some substances with certain adsorption, such as cellulose, chitin, etc.
FIG. 5 shows the cell morphology of Aspergillus niger A80 during growth on PDA solid medium. It can be seen that the conidia of Aspergillus niger A80 are spherical, brownish black, rapidly spread, and white at first, then turn into bright yellow to black thick velvet, and have colorless back or yellowish brown center. Conidial heads are in a brownish black radial shape, and conidial stalks are different in length. The top capsule is spherical and has double-layer small stems.
3.1.2ECP topography
Adding NaOH into the Aspergillus niger A80 fermentation liquor to adjust pH, performing ultrasonic treatment, performing high-speed centrifugation for 20min at 10000 rpm, taking supernatant, and slowly adding three times of volume of glacial ethanol. When the fermentation supernatant meets the ice ethanol, flocculent precipitate appears, which indicates that the ECP is rapidly condensed and separated out. With the continuous addition of the glacial ethanol, the ECP continuously condenses out, and aggregates settle to the bottom of the vessel. Placing the mixed solution in a refrigerator at 4 ℃ for standing for 24h, performing centrifugal separation at 5000 rpm to obtain an ECP sticky substance (figure 6), dehydrating the precipitate with absolute ethyl alcohol and acetone, repeatedly washing and centrifuging with 95% ethyl alcohol, dissolving the precipitate in deionized water again, transferring the precipitate into a dialysis bag with the relative molecular mass of 8000-14000 Da, placing the dialysis bag in deionized water for dialysis for 48h, and changing water every 6 h. After the dialysis is completed, the solution in the dialysis bag is collected, concentrated and freeze-dried, and finally the polymeric metabolite ECP of Aspergillus niger A80 is obtained (FIG. 7).
FIG. 6 shows crude ECP precipitated by glacial ethanol. Aspergillus niger A80 ECP is light yellow brown viscous solid, has high water content, is easily soluble in water, soluble in acidic and alkaline solution, and insoluble in organic solvent such as ethanol and acetone. The aqueous solution of aspergillus niger a80 ECP was a light tan transparent solution. Fig. 7 shows the ECP product obtained after dialysis and freeze-drying, and it can be seen that the dried ECP has a loose structure, a laminated structure, a porous surface and a very low powder density.
Scanning electron microscope observation (fig. 8-10) is carried out on the ECP sample after drying treatment, and the obvious difference of the surface appearance of the ECP products in different culture periods can be seen. 3d ECP was deposited in sheets (500 times), porous on the surface, very unsmooth (10000 times). This phenomenon is similar to the observation of Kumar c.g. et al, who finds ECP 471 as a porous structure under a scanning electron microscope with many small pores distributed on the surface. The scanning electron microscope with 500 times shows that the 6d ECP has an obvious linear main chain, each main chain unit forms a spherical, rod-shaped or branched side chain structure through different connection modes, a three-dimensional network structure with complicated branches is presented, and when the magnification of the electron microscope is increased to 10000 times, the surface of the 6d ECP is slightly concave-convex and is relatively flat. The electron microscope is magnified 500 times, the 10d ECP is a three-dimensional network structure formed by the cross-linking of a sheet structure, and is magnified 10000 times, so that the 10d ECP has a smooth surface, smooth edges and a large surface area.
3.1.3 specific surface area measurement of ECP
The specific surface area of the adsorbent has an important influence on the adsorption efficiency, so that the determination of the specific surface area of the adsorbent is of great significance for the study of the adsorption characteristics of the adsorbent on heavy metal ions. The method for measuring the specific surface area of the solid material mainly comprises a dye solution adsorption method, a BET low-temperature adsorption method, an electron microscopy method and the like.
FIG. 11 is an adsorption isotherm of Aspergillus niger A80 ECP for methylene blue at 25 deg.C, pH 7.0. As can be seen, the langmuir type is met. Langmuir-type adsorption theory is based on the following assumptions: 1) the dye is adsorbed by the adsorbent, namely the dye is adsorbed on a specific dyeing mat in a positioning way; 2) the dye mat has the characteristic of monomolecular adsorption, each dye mat only adsorbs one dye molecule, and if the dye mat is completely occupied, the dye mat is saturated; 3) the adsorbed dye molecules do not interact with each other and do not interfere with each other. As can be seen from the figure, the adsorption behavior of ECP to methylene blue at different periods is different, and the adsorption performance magnitude relationship is as follows: 6d ECP >3d ECP >10d ECP.
The data were fitted with a Languuir adsorption isotherm model (FIG. 12) to give 3d, 6d and 10d ECPs with methylene blue monolayer saturation adsorption amounts of 83.8, 105 and 69.7mg/g, respectively. The calculated specific surface areas of 3d, 6d and 10d ECP were 226, 336 and 221m, respectively2(ii) in terms of/g. The larger specific surface area of the Aspergillus niger A80 ECP is beneficial to the adsorption effect of the Aspergillus niger A80 ECP on heavy metal ions.
4 Aspergillus niger A80 ECP medium composition optimization
4.1 Single factor optimization experiment
4.2 selection of carbon sources in the Medium
When the influence of different carbon sources on the growth characteristics of the aspergillus niger A80 mycelial pellets and the yield of ECP is examined, lactose, sucrose, glucose and soluble starch are respectively used as single carbon sources, and ammonia chloride is used as a nitrogen source. A number of 250mL Erlenmeyer flasks were charged with 150mL carbon-free fermentation medium. Different carbon sources were added to the samples with a carbon concentration of 42 g/L. After the culture medium is sterilized at 121 ℃ for 20min, 1.5mL of spore suspension (107 spores/mL) is inoculated and placed in a constant-temperature shaking incubator at 150rpm and 25 ℃ for culture. Mycelial pellet diameter and dry weight, ECP yield and TOC concentration were measured at 1, 2, 3, 4, 6, 8 and 10d, respectively, with 3 replicates for each experiment.
4.3 selection of Nitrogen sources in the Medium
When the influence of different nitrogen sources on the growth characteristics of the aspergillus niger A80 mycelial pellets and the yield of ECP is examined, ammonium sulfate, ammonium chloride, beef extract, peptone and yeast extract are respectively used as single nitrogen sources, and sucrose is used as a carbon source. A number of 250mL Erlenmeyer flasks were each charged with 150mL of nitrogen-free fermentation medium. With the nitrogen concentration of 1g/L as a standard, different nitrogen sources are respectively added. The culture medium is sterilized at 121 ℃ for 20min, and after cooling at room temperature, 1.5mL of spore suspension (107 spores/mL) is inoculated and placed in a constant-temperature shaking incubator at 150rpm and 25 ℃ for culture. Mycelial pellet diameter and dry weight, ECP yield and TOC concentration were measured at 1, 2, 3, 4, 6, 8 and 10d of culture, respectively, and the experiment was repeated 3 times.
4.4 mycelium pellet diameter; randomly selecting 20 mycelium pellets, absorbing surface moisture by using absorbent paper, arranging the mycelium pellets into a straight line, measuring the total length of the mycelium pellets, and calculating an average value.
4.5 mycelium pellet dry weight; randomly selecting 20 mycelium pellets, sucking surface water by using absorbent paper, placing the culture dish carrying the mycelium pellets in a 95 ℃ oven to be dried to constant weight, and measuring the dry weight of the mycelium pellets.
4.6 mycelium pellet growth state parameter (H); the mycelium pellet has huge specific surface and strong adsorption efficiency, and the parameter (H) for evaluating the growth state of the mycelium pellet is provided by referring to the porosity of the activated carbon.
H=G/(D)3(2-3);
In the formula: h-growth state parameter of mycelium pellet, kg/L; g-mean dry weight of mycelium pellets, mg; d-mean diameter of mycelium pellet, mm.
And H, characterizing the mycelium dry weight of the mycelium pellet in unit volume, namely the mycelium density. And measuring the diameter and dry weight change of the mycelium pellet in the culture medium with different carbon sources and nitrogen sources, and evaluating the growth state of the mycelium pellet according to the change condition of H, thereby determining the optimal carbon source and nitrogen source of the culture medium.
4.7 center combination experimental design
Four important factors were further studied using a central combinatorial experimental design: the effect of carbon source dosage (X1), nitrogen source dosage (X2), medium pH (X3) and culture period (X4) on ECP production. Firstly, a Central Composite Design (CCD) is adopted to design a Central composite design of 30 tests, wherein the Central composite design consists of 16 full-factor tests, 8 axial points, 2 software-generated Central points and 4 increased Central points. And fitting and regression analysis are carried out on the experimental result by adopting a response surface model with a 4-factor 5 level to obtain a quadratic polynomial, and the relation between the significant factors influencing the ECP production of the Aspergillus niger A80 and the response value can be described. The partial experimental Design and data analysis was done by Design Expert 8.0.6 software. Meanwhile, the three-dimensional response surface graph is used for explaining the influence of the influencing factors on the response values and the interaction influence among the influencing factors.
4.8 model fitting and Experimental validation
And fitting the experimental data by using a response surface model to obtain each parameter in the model. And performing simple character analysis on the multivariate function to determine the extreme point and the value of the independent variable corresponding to the extreme point. And (5) carrying out experiments according to the most conditional conditions, verifying the reliability of the model, and determining the final optimization result.
5 Aspergillus niger A80 ECP culture medium component optimization
5.1 introduction to
The mass production of microbial ECP usually adopts a fermentation production method, and in order to improve the yield of ECP in the fermentation process, two main ways exist: 1) improving the ECP biosynthesis capacity of the ECP producing strain by utilizing a gene technology; 2) the fermentation process is optimized. In fermentation processes, the media raw material costs make up a considerable proportion of the total cost. Therefore, to reduce the overall production cost while increasing the ECP yield, optimization of the composition of the medium is required. The research aims to discuss the influence of different nutritional conditions on growth of the aspergillus niger A80 mycelial pellets and ECP secretion, optimize the components of the culture medium and improve the ECP yield so as to realize development and utilization of aspergillus niger A80.
5.2 Single factor experiment of fermentation conditions
5.3 selection of carbon sources in the Medium
The diameter, dry weight and ECP yield of the Aspergillus niger A80 mycelial pellets were determined at different culture periods using sucrose, glucose, lactose and soluble starch as single carbon sources for Aspergillus niger fermentation, respectively. As shown in Table 2, the average diameter, average dry weight and total dry weight of the mycelium pellets showed a tendency to increase gradually with time under different carbon sources, indicating that Aspergillus niger A80 can grow in a medium containing sucrose, glucose, lactose and soluble starch as single carbon sources. Wherein the growth amount of the mycelium pellets in a culture medium taking sucrose as a single carbon source is the highest, and the total dry weight of the mycelium pellets at 10d is 9.92 g/L; in a culture medium with lactose and glucose as single carbon sources, the total dry weight of the mycelium pellet of 10d is 6.24g/L and 3.37g/L respectively; in a culture medium using soluble starch as a single carbon source, the thalli grow slowly, the growth amount is low, the total dry weight of 10d mycelium pellets is only 1.68g/L, and the soluble starch is not easy to be utilized by the strain. In addition, when lactose is used as a carbon source, the growth state parameter (H) value of the mycelium pellet is the largest (0.98g/L), and the time required for reaching the maximum H value is the shortest, which indicates that the growth speed of the mycelium pellet is the fastest under the condition that lactose is used as the carbon source.
As can be seen from Table 3, when the strain uses lactose and glucose as single carbon sources, a large amount of ECP can be synthesized, and the ECP yield is 2.33g/L and 2.11g/L respectively; while the yield of ECP was lower when sucrose and soluble starch were used as carbon sources, 1.51g/L and 1.14g/L, respectively. From the comprehensive consideration of the growing speed of mycelium pellets and the yield of ECP, lactose can be selected as a long-acting carbon source in the Aspergillus niger A80 fermentation liquid process, and sucrose can be selected as a short-acting carbon source in the Aspergillus niger A80 fermentation liquid process from the consideration of raw material sources and industrial application cost.
TABLE 2 influence of carbon sources on mycelial sphere diameter, dry weight and State parameters
Figure BDA0002914047480000141
TABLE 3 influence of carbon sources on ECP production and pH of fermentation broths
Figure BDA0002914047480000142
5.4 selection of Nitrogen sources in the Medium
The diameter, dry weight and ECP yield of Aspergillus niger A80 mycelium pellet were measured at different culture periods using ammonium sulfate, ammonium chloride, beef extract, peptone and yeast extract as single nitrogen sources, respectively. As shown in Table 4, Aspergillus niger A80 was able to grow in a medium containing ammonium sulfate, ammonium chloride, beef extract, peptone and yeast extract as single nitrogen sources. Wherein the growth amount of the mycelium pellet is the highest in a culture medium taking yeast extract as a single nitrogen source, and the total dry weight of the mycelium pellet at 10 days is 9.58 g/L; in a culture medium with peptone, ammonium chloride and beef extract as single nitrogen sources, the total dry weight of the mycelium pellets of 10 days is 7.51g/L, 7.04g/L and 6.58g/L respectively; in a culture medium using ammonium sulfate as a single nitrogen source, the thalli grow slowly, the growth amount is low, the total dry weight of 10d mycelium pellets is only 3.22g/L, and the ammonium sulfate is not easy to be utilized by the strain. In addition, when the yeast extract is used as the nitrogen source, the growth state parameter (H) value of the mycelium pellet is the largest (0.50g/L), and the time required for reaching the maximum H value is the shortest, which shows that the growth speed of the mycelium pellet is the fastest under the condition that the yeast extract is used as the single nitrogen source.
TABLE 4 influence of nitrogen sources on the diameter, dry weight and state parameters of mycelial pellets
Figure BDA0002914047480000151
As can be seen from Table 5, when the strain uses yeast extract as a single nitrogen source, a large amount of ECP can be synthesized, and the yield of ECP is 2.55 g/L; when ammonium sulfate, ammonium chloride, beef extract and peptone are used as single nitrogen sources, the ECP yield is equivalent and is respectively 1.49g/L, 1.48g/L, 1.37g/L and 1.58 g/L. Considering the growing speed of mycelium pellets and the yield of ECP, and the source of raw materials and the cost of industrial application, it is more appropriate to select yeast extract as the nitrogen source of the culture medium in the process of Aspergillus niger A80 fermentation liquid.
TABLE 5 influence of nitrogen source on ECP production and pH of fermentation broth
Figure BDA0002914047480000161
5.3 center combination design
The effect of four factors, lactose dosage (X1), yeast extract dosage (X2), medium pH (X3) and culture period (X4), on ECP yield was studied at five levels in the range of-2 to +2 using Central Composite Design (CCD) [108], and the factors and levels of Central composite design are shown in table 6. The experiment was performed according to the central combination design table of 30 experiments consisting of 16 full factor experiments, 8 axial points, 2 software generated central points and 4 added central points, and the design and response values of the central combination experiments are shown in table 7.
TABLE 6 center combination design factors and levels
Figure BDA0002914047480000162
TABLE 7 center combination experimental design and results
Figure BDA0002914047480000163
Figure BDA0002914047480000171
And performing regression analysis on the experimental result by adopting a four-factor five-level response surface model, and fitting the experimental data to describe the relationship between the significant factors influencing the ECP production of the Aspergillus niger A80 and the response value.
5.4 response surface analysis
FIGS. 13-15 are three-dimensional response surface plots of pairwise variables versus ECP yield, reflecting the effect of each impact factor and its interaction on the response values.
The optimization result shows that when the lactose adding amount, the yeast extract adding amount, the culture medium pH and the culture period are respectively 120g/L, 2.0g/L, 6.5 h and 116h, the model predicts that the ECP yield is the highest and reaches 339TOC mg/L.
5.5 model fitting and Experimental validation
And carrying out experiments according to the optimal conditions obtained by the response surface to verify the reliability of the model and determine the optimized result. The yield of the optimized ECP is 324mg/L, and the error of the model fitting result is within 10 percent, which indicates that the obtained model is reliable.
Summary of the invention
(1) Through single factor experiments of an optimal carbon source, the aspergillus niger A80 can grow well in a culture medium with glucose, sucrose and lactose as single carbon sources, and soluble starch is not easy to be utilized by the strain. When sucrose is used as a single carbon source, the growth amount of the mycelium pellets is the highest; when lactose is used as a carbon source, the growth speed of the mycelium pellets is the fastest. From the comprehensive consideration of the growing speed of mycelium pellets and the yield of ECP, lactose can be selected as a long-acting carbon source in the fermentation liquid process of Aspergillus niger A80, and sucrose can be selected as a short-acting carbon source from the consideration of raw material sources and industrial cost.
(2) Through single factor experiments of an optimal nitrogen source, the aspergillus niger A80 can grow in a culture medium with ammonium sulfate, ammonium chloride, beef extract, peptone and yeast extract as single nitrogen sources. When the yeast extract is used as a single nitrogen source, the growth amount of the mycelium pellets is the highest, the growth speed of the mycelium pellets is the fastest, and the yeast extract is selected as the nitrogen source of the culture medium in the process of the Aspergillus niger A80 fermentation liquid.
(3) By applying the central combination design, 4 influence factors are optimized through 30 experiments. The optimization result shows that when the lactose adding amount, the yeast extract adding amount, the culture medium pH and the culture period are respectively 120g/L, 2.0g/L, 6.5 h and 116h, the model predicts that the ECP yield is the highest and reaches 339TOC mg/L. The yield of the optimized ECP is 324mg/L, and the error of the model fitting result is within 10 percent, which indicates that the obtained model is reliable.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. An optimized culture medium for Aspergillus niger to produce extracellular polymers, the optimized culture medium for Aspergillus niger to produce extracellular polymers comprising: plate culture medium-PDA culture medium, fermentation culture medium;
plate medium-PDA medium: 200g/L of potato, 15-20 g/L of agar, 20g/L of glucose, natural pH and autoclaving at 121 ℃ for 20 min;
fermentation medium: NaNO31.5 g/L,KH2PO40.5 g/L,KCl 0.025g/L,MgSO4·7H20.025g/L of O, 100g/L of sucrose, 1.6 of yeast extract, natural pH and autoclaving at 121 ℃ for 20 min.
2. The optimized culture medium for producing extracellular polymeric substances by Aspergillus niger according to claim 1, wherein potato is peeled and washed in the plate culture medium, PDA culture medium, 200g is weighed, cut into small pieces, boiled in water until the potato is softened, filtered by eight layers of gauze, the calculated amount of agar and glucose are added to the filtrate, the mixture is mixed uniformly and supplemented to 1000mL by distilled water, sterilized at 121 ℃ for 20min, and made into slant or plate, and the culture medium is used for strain preservation.
3. An optimized culture method for producing extracellular polymeric substances by Aspergillus niger according to any one of claims 1-2, comprising:
step one, selecting a strain of Aspergillus niger A80 with high ECP yield as an experimental material, preparing spore suspension, and carrying out strain fermentation culture;
extracting, purifying and analyzing the quantity, chemical components and structures of ECP generated in different growth stages, and exploring the complexation and mechanism of ECP and heavy metal ions;
and step three, optimizing the culture conditions for producing the ECP by the A.niger A80 by a response surface method.
4. The optimal culture method for producing extracellular polymeric substances by Aspergillus niger according to claim 3, wherein in the first step, the spore suspension is prepared by:
washing bacterial colony on the surface of the plate culture medium with quantitative sterile water on a sterile operating platform, transferring the eluted spores into a sterilized conical flask, repeating the steps for several times, adding 0.02% w/v sodium dodecyl sulfate, and shaking uniformly to obtain a high-concentration spore suspension with the spore concentration of 107one/mL.
5. The optimal culture method for producing extracellular polymeric substances by Aspergillus niger according to claim 3, wherein in the first step, the strain fermentation culture is as follows: 300mL of fermentation medium was placed in a 500mL conical flask, 3mL of spore suspension was inoculated after sterilization, and the mixture was placed at 25 ℃ in a shaker at 150rpm for fermentation for a predetermined period of time.
6. The optimal culture method for producing extracellular polymeric substance by Aspergillus niger according to claim 3, wherein in the second step, the extraction method of crude ECP comprises: adding strong alkali sodium hydroxide into the thallus fermentation liquor to increase the pH value of the system.
7. The optimal culture method for producing extracellular polymeric substance by Aspergillus niger according to claim 3, wherein the second step comprises the step of purifying ECP by: concentrating the supernatant, removing protein by Seveag method, sequentially adding chloroform and n-butanol into the supernatant, shaking the mixed solution sufficiently, centrifuging at 8000rpm for 10min at 4 deg.C, removing middle denatured protein and lower layer chloroform, pouring out the supernatant, and repeating for several times until no obvious denatured protein appears in the middle;
the ECP solution after deproteinization treatment is subjected to fractional precipitation, freezing and centrifugation by adopting ethanol to remove nucleic acid, firstly, 25% of glacial ethanol is used for precipitation to remove nucleic acid, supernatant fluid is collected by centrifugation, 75% of glacial ethanol is reprecipitated, precipitate is sequentially washed by absolute ethyl alcohol and acetone and then dissolved in distilled water again, the precipitate is placed in a dialysis bag of 8000-14000 Da at the temperature of 4 ℃ for dialysis for 48h, and the dialysis solution in dialysis is collected and freeze-dried to obtain the pure ECP.
8. The culture method for optimizing the production of extracellular polymeric substance by Aspergillus niger according to claim 7, wherein the ratio of chloroform to n-butanol is 5:1, V/V.
9. The optimal culture method for producing extracellular polymeric substance by Aspergillus niger according to claim 7, wherein the conditions for removing nucleic acid by 25% ethanol precipitation are as follows: 24 hours at 4 ℃; the conditions for 75% glacial ethanol reprecipitation were: 4 ℃ and 24 h.
10. The optimal culture method for producing extracellular polymeric substance by Aspergillus niger according to claim 3, wherein the third step comprises the following specific processes: fitting the experimental data by using a response surface model to obtain each parameter in the model;
performing simple character analysis on the multivariate function to determine an extreme point and a value of an independent variable corresponding to the extreme point;
and carrying out experiments according to the optimal conditions, verifying the reliability of the model, and determining the final optimization result.
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CN1759186A (en) * 2003-01-09 2006-04-12 健泰科生物技术公司 Purification of polypeptides
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CN1759186A (en) * 2003-01-09 2006-04-12 健泰科生物技术公司 Purification of polypeptides
US20140073026A1 (en) * 2008-09-15 2014-03-13 The Goodyear Tire & Rubber Company Systems using cell culture for production of isoprene
CN106190871A (en) * 2016-09-18 2016-12-07 南京农业大学 A kind of method that compound thread fungal organism drip leaching with straw as carbon source processes heavy-metal contaminated soil

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